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RESULTS OF THE STUDY OF BIOLOGICAL AND MOLECULAR GENETIC PROPERTIES OF BIFIDOBACTERIUM STRAINS - PROBIOTIC PRODUCERS
A.G. Tochilina1'3*, I.V. Belova1'3, I.V. Soloveva1, T.N. Ilyicheva2, D.B. Gelashvili4, V.A. Zhirnov1 S.B. Molodtsova1
1 Academician I.N. Blokhina Nizhny Novgorod Scientific Research Institute of Epidemiology and Microbiology (Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing), 71 Malaya Yamskaya St., Nizhny Novgorod, 603950, Russia;
2 State Research Center of Virology and Biotechnology «Vector» of Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, Koltsovo, Novosibirsk region, 630559, Russia;
3 Privolzhsky Research Medical University, 10/1 Minin and Pozharsky Sq., Nizhny Novgorod, 603950, Russia;
4 Lobachevsky State University of Nizhny Novgorod, 23 Prospekt Gagarina, Nizhny Novgorod, 603022, Russia.
* Corresponding author: lab-lb@yandex.ru
Abstract. For a long time, the strains Bifidobacterium bifidum 1, Bifidobacterium bifidum 791, and Bifidobacterium longum 379 have been successfully used as probiotic producers; the conclusions about the possibility of their use, their role and functions in the human body were made empirically based on indirect signs. At present, high-tech equipment and modern research methods allow to conduct a complete study of the properties and characterization of strains; it is regulated by modern regulatory documents. In our work we applied the classical bacteriological method, MALDI TOF mass spectrometry method, NGS whole genome sequencing. The analysis of 27 clones of the strains revealed that they have typical morphological properties and stable biochemical profiles; the lists of ionized protein masses (so-called «strain markers») were determined. The whole genomes of strains deposited by us in GenBank in 2015-2017 were analyzed for the first time; we found that they do not contain pathogenicity genes, integrated plasmids, determinants of transmissible antibiotic resistance, and belong to phylogenetic clusters formed by probiotic-producing strains. It was shown that B. bifidum 1 and B. bifidum 791 have a pronounced ability to consume glycans of intestinal mucus, while
B. longum 379 can efficiently consume plant glycans. The genomes of B. bifidum 791 and B. longum 379 strains contain genes for the synthesis of the most important neurometabolites of tryptophan and folic acid; the genome of B. bifidum 791 strain contains genes for the synthesis of lasso peptide and flavucin, class I lantibiotics with a wide spectrum of antimicrobial and antiviral activity.
Keywords: Bifidobacterium, producer strains, probiotics, whole genome sequencing, MALDI TOF mass spectrometry, bacteriocins.
List of Abbreviations
BLAST - basic local alignment search tool (a family of computer programs used to search for homologues of proteins or nucleic acids) CDS - coding sequence EC - enzyme classification GH - glycosil hydrolase MALDI - matrix-assisted laser desorp-tion/ionization
MSP - main spectra
RAST - rapid annotation using subsystems technology
SNP - single nucleotide polymorphism TOF - time of flight a-CHCA - a-Cyano-4-hydroxycinnamic acid
PM - pathogenic microorganisms
OM - opportunistic pathogens
SCFAs - short-chain fatty acids
Introduction
For a long time, strains Bifidobacterium bifidum 1, Bifidobacterium bifidum 791, and Bifidobacterium longum 379 have been successfully used as probiotic producers (Belova et al., 2017; Tochilina et al., 2016; Belova et al., 2016; Soloveva et al., 2012). The conclusions on the possibility of their use as well as their role and functions in the human body were made empirically based on indirect signs. At present, the availability of high-tech equipment and modern research methods, such as MALDI TOF mass spectrometry and whole genome sequencing, allows to conduct a complete study
of the properties and characterization of strains. In this regard, according to modern regulatory documents, the characteristics of producer strains should be supplemented with data on the stability of their properties, the absence of pathogenicity genes, integrated plasmids, transmissible antibiotic resistance genes, genetically determined metabolic potential, and the ability to synthesize biologically active substances (Guidelines for the control of biological and microbiological factors, 2011; Guidelines for the sanitary and epidemiological assessment of the safety and functional potential of probiotic microorganisms, 2010).
The knowledge about the characteristics of the metabolism of probiotic-producing strains, the spectrum of their enzymes and preferred sources of nutrition within the framework of evidence-based medicine contribute to the development of a personalized approach to prescribing probiotic therapy, taking into account the properties of each strain included in the drug or product, in a particular pathology accompanied by certain metabolic disorders associated, among other things, with changes in the structure and functions of the patient's microbiome. The presence of a wide range of bacterial hy-drolases contributes to the most efficient absorption of food by the macroorganism, including complex carbohydrates of plant origin with the formation of a range of valuable metabolites such as short-chain fatty acids (SCFAs), which have a complex positive effect on human health (Kornienko, 2016; Oleskin & Shenderov, 2016). The ability of producer strains to synthesize neurotransmitters and their precursors affects the concentration of the most important neurotransmitters in the human body, the lack of which causes brain disorders and mental changes (Oleskin & Shenderov, 2019). A strain-specific property is the synthesis of bacteriocins - antimicrobial peptides that cause a pronounced antibacterial and antiviral activity of microorganisms, which allows probiotic strains to take part in the formation of «joint immunity», that is, to inhibit and eliminate opportunistic and pathogenic flora together with macroorganism's immunity factors (Oleskin & Shenderov, 2019; Arena et al., 2018; Kim et al.,
2018; Lei et al., 2016; Majamaa et al., 1995; Soloveva et al., 2021).
In this regard, there is no doubt about the relevance of the in-depth studies of the biological and molecular genetic properties of probiotic strains of bifidobacteria used for the production of medicines and foodstuffs in terms of assessing their probiotic and metabolic potential, the ability to synthesize glycosyl hydrolases, neurotransmitters, and bacteriocins.
The purpose of the study is an in-depth study of the biological properties and analysis of the genome features of producing probiotic strains Bifidobacterium bifidum 1, Bifidobacterium bi-fidum 791, Bifidobacterium longum 379.
To achieve this goal, it was necessary to solve the following tasks: to conduct profiling of bacterial proteins of strains using the Bruker MALDI TOF mass spectrometer and Biotyper software to identify individual characteristics; to evaluate the stability of the biochemical properties of strains; to analyze whole genome sequences of strains for pathogenicity determinants, antibiotic resistance genes, and integrated plasmids; evaluate the genetically determined metabolic potential of strains, their ability to synthesize glycosyl hydrolases, neurotransmitters, and bacteriocins; to determine the phylogenetic relationships of the studied strains with other strains of these species deposited in GenBank, including probiotic-producing strains; to determine characteristics of pan- and core genomes of all strains of B. bifidum and B. longum deposited in GenBank; to conduct a comparative analysis of the similarity of the amino acid sequences of proteins determined by the core genome genes of the studied strains and other strains of these species deposited in GenBank, including probiotic-producing strains.
Materials and Methods
The study was approved by the Ethics Committee of Privolzhsky Research Medical University.
Strains used in the study
The strains B. bifidum 1 (10 clones = ampoules), B. bifidum 791 (10 clones = ampoules),
B. longum 379 (10 clones = ampoules) were used in the study.
B. bifidum 1 is a production strain used in the production of bifid-containing probiotics registered in the Russian Federation (GPM. 1.7.1.0003.15) (General Pharmacopoeia Monograph, 2015), deposited in the State collection of pathogenic microorganisms (collection number of the strain - No. 900791) and the State collection of normal microflora microorganisms of G.N. Gabrichevsky Moscow Scientific Research Institute of Epidemiology and Microbiology (Federal Service for Surveillance on Consumer Rights Protection and Human Well-being). Academician I.N. Blokhina Nizhny Novgorod Scientific Research Institute of Epidemiology and Microbiology (formerly -Gorky Scientific Research Institute of Epidemiology and Microbiology) received the strain in the 1970s during the joint work with G.N. Gabrichevsky Moscow Scientific Research Institute of Epidemiology and Microbiology on the production of Bifidumbacterin.
B. bifidum 791 is a production strain used in the production of bifid-containing probiotics registered in the Russian Federation (GPM.1.7.1.0003.15) (General Pharmacopoeia Monograph, 2015), deposited in the Russian national collection of industrial microorganisms (VKPM) (deposit number - No. B-3300) and the State collection of normal microflora microorganisms of G.N. Gabrichevsky Moscow Scientific Research Institute of Epidemiology and Microbiology. The strain was transferred to Academician I.N. Blokhina Nizhny Novgorod Scientific Research Institute of Epidemiology and Microbiology (formerly -Gorky Scientific Research Institute of Epidemiology and Microbiology) together with the B. bifidum 1 strain for the production of fermented milk Bifilact.
B. longum 379 is a production strain used in the production of bifid-containing probiotics registered in the Russian Federation (GPM. 1.7.1.0003.15) (General Pharmacopoeia Monograph, 2015), the Russian national collection of industrial microorganisms (VKPM) (deposit number - No. B-2000) and the State collection of normal microflora microorganisms of G.N.
Gabrichevsky Moscow Scientific Research Institute of Epidemiology and Microbiology. It was transferred to Academician I.N. Blokhina Nizhny Novgorod Scientific Research Institute of Epidemiology and Microbiology (formerly -Gorky Scientific Research Institute of Epidemiology and Microbiology) in 1989 during the joint work.
Cultivation of strains
To restore and sieve strains of Bifidobacterium, after opening the ampoule, the lyophilic mass was poured with 2 ml of bifidum medium (nutrient medium for the isolation and cultivation of bifidobacteria, dry, Obolensk), transferred to a sterile test tube with 9 ml of bifidum medium and incubated at 38 ± 1°C for 72 hours (the first generation of the strain). After 2 days, 2 ml of the first generation were inoculated into 50 ml of bifidum medium and incubated at 38 ± 1°C for 72 hours (the second generation of the strain).
Next, 1 ml of the second generation of the strain was titered in bifidum medium poured in a high column into 9 ml test tubes to a dilution of 10-11. Dilutions of 10-6, 10-7, 10-8, 10-9 were used to conduct 1 ml inoculations onto a plate medium for cultivating bifidobacteria (nutrient medium for determining and counting bifidobacteria, dry, State Research Center for Applied Microbiology and Biotechnology, Obolensk) and incubated for 72 hours at 38 ± 1°C under anaerobic conditions using GasPak Anaerobe Gas Generating Pouch System with Indicator, USA.
The morphology of grown colonies of microorganisms was evaluated; 10-12 colonies of each strain were selected for the next stage of the study: a sample from each colony was applied to 3 target cells for subsequent mass spectrometry, and the remainder was seeded in a bifidum medium for subsequent biochemical identification. Microscopy of Gram-stained smears was performed using a Micros MC500 laboratory microscope (Micros, Austria). Cultures that had Score values of 2.100 or more according to the results of mass spec-trometry were selected for biochemical identification.
Strain identification and bacterial protein profiling
Identification and profiling of bacterial proteins of strains weree carried out using an Autoflex speed LRF time-of-flight MALDI mass spectrometer (Bruker Daltonics, Germany) equipped with a modified solid-state laser. All measurements were performed in a linear mode, detecting positive ions. To accumulate mass spectra, the laser radiation power was set at the level of the minimum threshold value sufficient for the desorption-ionization of the sample. The mass spectrometer parameters were optimized for the m/z range from 2000 to 20000. To obtain mass spectra suitable for identification, the following parameters were set in the device settings: summation of 10 series of spectra with 50 laser pulses each. External calibration was performed using a bacterial test standard (Bruker Daltonics, Germany) with a-cyano-4-hy-droxycinnamic acid (a-CHCA) as a matrix. Sample preparation of cultures of the studied microorganisms was performed according to the standard operating protocol «Formic acid extraction» (Chebotar et al., 2018). Identification, recording, processing, and analysis of mass spectra were carried out using the Bio-Typer RTC program. The accuracy of identification was judged by the value of the coefficient of coincidence (Score values: 2.0003.000 - identification to the species; 1.9991.700 - identification to the genus; 1.699-0 -identification failed) and the value of the categories (A - reliable identification up to the specie level, B - reliable identification up to the genus level, C - unreliable result).
The flexAnalysis program was used for profiling bacterial proteins, and the BioTyper MSP Dendrogram Creation Standard Method package included in the BioTyper 3.0 software and hardware complex was used to create an MSP dendrogram. Mass spectra from the Bruker database and the database of Academician I.N. Blokhina Nizhny Novgorod Scientific Research Institute of Epidemiology and Microbiology served as reference mass spectra. A total of 374 clones of three strains of bifidobacteria were identified.
Study of biochemical properties of strains
The biochemical properties of the strains were studied using standard API20A biochemical test systems (BioMerueux, France). To study the biochemical activity of bifidobacteria strains, 9 clones of each strain with a high Score were selected from cultures previously analyzed by mass spectrometry. Cultivation, sample preparation, identification of microorganisms, and interpretation of the obtained results were carried out according to the manufacturer's instructions; a total of 27 cultures were studied.
Whole genome sequencing
For the whole genome study, one culture of each strain with the highest Score and the studied biochemical profile was selected. Genomic DNA was isolated using a commercial kit QI-Aamp DNA Mini Kit «QIAGEN» (Germany); fragmentation was performed using a Covaris E210 ultrasonic fragmentation system «Applied Biosystems» (USA) according to the manufacturer's instructions. The mixture was purified, and fragments of 200-700 bp were selected using magnetic particles Agencourt AM-Pure beads «Beckman Coulter» (USA) and NEBNext Sizing Buffer «New England Biolabs» (USA). Libraries were prepared using the kit TrueSeq «Illumina Inc.» (USA); sequencing was performed on the platform MiSeq «Illumina Inc.» (USA). The original reads were processed by the Trimmomatic utility with standard parameters for Illumina. The processed reads were used for de novo genome assembly using the Spades, MIRA 4.0, and New-bler 2.6 programs.
Genome annotation
Genomes were annotated using the Prokka utility v. 1.11 (Seemann, 2014) and the RAST genomic server (http://rast.nmpdr.org). The search for determinants of antibiotic resistance and pathogenicity was carried out using software products presented on the website of the Center for Genomic Epidemiology (www.cge.cbs.dk): ResFinder 2.0, Pathogen Finder and PlasmidFinder (Seemann, 2014; Zankari et al., 2012; Cosentino et el., 2013;
Carattoli et al., 2014). The Bagel 4 program (van Heel et al., 2018) was used to detect the genetic determinants responsible for the production of bacteriocins. Key enzymes responsible for the synthesis of neurometabolites were searched using the RAST genomic server (http://rast.nmpdr.org) (Aziz, 2008) and scientific literature data (Rossi et al., 2011; Gabris et al., 2015).
Construction of genomic maps
Genomic maps were constructed using the Proksee service (https://proksee.ca); hierarchical structures (dendrograms) constructed on the basis of SNP analysis of whole genome sequences of strains and describing their phyloge-netic position were borrowed from the NCBI website.
Analysis of pan- and core genome of strains
To analyze the pan- and core genome, we used the PATO R package (Fernandez-de-Bobadilla et al., 2021), designed to analyze pangenomes of the same or different species (intraspecific/interspecific). PATO allows the analysis of population structure, phylogenetics, and horizontal gene transfer, using in each case a core genome (a set of genes common to all genomes), an accessory genome, or the entire genome. The files with format fna (fasta) containing amino acid sequences for the annotated proteins of the studied strains were used; next, redundancy-based filtering was performed, and identical strains (99.99% identity) were excluded from the analysis. The construction of ortholog clusters was carried out using MMSeq2 with standard parameters.
Study of the genetic diversity of strains
To study the level of genetic diversity of the strains, matrices of changes were constructed based on the results of the alignment of the amino acid sequences of the core genome proteins of the strains. The matrix was normalized by the total genome length (SNP/Megabase), then hierarchical clustering was performed. Matrix visualization is presented as a heat map; the color on the heat map represents the number of changes in the proteins of the core genome
of a particular strain and varies from dark blue (0 changes) to red (30000 changes). Analysis of the metabolic potential of the strains was performed using the RAST genomic server; annotation of glycosyl hydrolases was carried out using the dbCAN2 web service, which allows automated annotation of carbohydrate-active enzymes (Zhang et al., 2018).
Results
Morphological, tinctorial, and cultural properties of the studied strains of bifidobacte-ria
When cultivating on a plate medium (nutrient medium for determining and counting bifidobacteria, State Research Center for Applied Microbiology and Biotechnology, Ob-olensk) under anaerobic conditions, after 72 h of incubation at (38 ± 1) °C, B. bifidum 1 strain forms white, flat, with scalloped edge colonies with a convex center or white, flat, shiny colonies on the surface of the medium; B. bifidum 791 strain forms white, shiny colonies with a smooth edge, and B. longum 379 - convex, with even edges, white, shiny colonies with a smooth edge and creamy consistency. On a liquid medium (Bifidum medium, Obolensk), all strains were characterized by near tube bottom growth and colonies of «nail-shaped» form in the depth of the medium; Gram-stained smears showed gram-positive polymorphic rods with uneven edges and bifurcations, arranged as single cells or clusters in the form of Chinese characters.
Thus, it was found that despite long-term cultivation in laboratory conditions, all the studied strains have stable morphological, tinctorial, and cultural properties.
Results of strain identification and bacterial protein profiling using MALDI TOF mass spectrometry
Identification and analysis of mass spectra of 374 cultures of bifidobacteria were performed.
Strain identification using mass spectrome-try was carried out using formic acid protein extraction according to the standard operating protocol, and the results are presented in Table 1.
Table 1
Results of identification of the studied strains of microorganisms using MALDI-TOF mass spectrometry
No Strain Number of cultures identified Mass spectrometry result
Microorganism Score values, category
1 B.bifidum 1 132 B.bifidum From 1.878 B to 2.375 A
2 B. bifidum 791 118 B.bifidum From 1.935 B to 2.472 A
3 B. longum 379 124 B. longum From 1.848 B to 2.339 A
Fig. 1. MALDI mass spectra of strains of the genus Bifidobacterium using a-CHCA matrix
The MALDI TOF mass spectrometry principle is based on the measurement of the mass of an ionized substance. During MALDI ionization, singly charged ions are formed, i.e., one ion corresponds to one protein, so the method allows to identify a unique set of proteins for each of the studied microorganisms. When forming the mass spectrum, the resulting ions line up in ascending order of mass, while the intensity (height) of the peaks is not considered. When identifying microorganisms, the entire set of peaks is considered, not individual peaks.
Individual mass spectra were obtained for each bifidobacterial strain under study; typical MALDI mass spectra are shown in Fig. 1. Although the mass spectra were taken in the range of 2000-20000 m/z, the area from 2000 to 10000 m/z is visually the most informative.
The results of direct protein profiling can be obtained not only in graphical form (mass spectra) but also in tabular form (mass lists), which makes it possible to analyze all the presented peaks. Tables of the most reproducible mass peaks of three bifidobacteria strains were compiled as a result of this work (Table 2).
Table 2
List of the most reproducible mass peaks of the studied strains
B. bifidum 1 B. bifidum 791 B. longum 379
m/z m/z m/z
13183.536
13124.124
12027.446 12027.564
11977.643
10447.332 10445.155
10357.955 10356.349 10358.708
10139.258 10138.777
10121.511
9842.616 9841.157
9831.527
9505.717 9502.255
9261.566
9288.645 9285.021
8928.336 8927.405
8790.287
8716.231
8316.336
8193.591
7954.878 7954.121 7952.583
7615.687 7617.355
6797.350 6797.271 6797.925
6567.926
6544.080 6546.398
6390.823 6390.442
6305.199
6014.847 6014.701
5991.202
5180.380 5179.643 5180.542
4921.924 4921.951
4916.680
4631.920
4395.924
4374.763 4374.587 4373.791
3978.057 3978.322 3977.529
3398.104 3399.177
3283.846
3273.694 3273.416
3153.084
2304.624 2304.219
2186.407 2186.754
A comparative analysis of 374 obtained mass spectra revealed that a number of peaks are common for all three strains - corresponding to m/z values of 10357, 7954, 6797, 5180, 4374, 3978. The lists of ionized protein masses,
the so-called «strain markers», were determined based on the analysis of the mass spectra of 374 strain clones. The B. bifidum 791 strain is characterized by peaks corresponding to the following masses m/z: 12027, 10445, 10138,
9841, 9502, 9285, 8927, 6546, 6390, 6014, 4921, 3399, 3273, 2304, 2186; for strain B. bifidum 1 the following peaks are specific: 13183, 10121, 8716; for strain B. longum 379: 13124, 11977, 9831, 9261, 8790, 8316, 8193, 6567, 6305, 5991, 4916, 4631, 4395, 3283, 3153.
Results of studying the biochemical properties of strains
The studied bifidobacteria are strains-producers of probiotic medicines and foodstuffs. In accordance with the requirements of regulatory documents, their biochemical properties must be stable regardless of the duration of storage and cultivation methods and are subject to constant laboratory control.
To study the biochemical activity, 27 pure cultures of bifidobacteria with a high Score were selected from the strains analyzed earlier by mass spectrometry (Table 3).
9 clones of B. bifidum 1, B. bifidum 791, and B. longum 379 strains with Score values from 2.19 to 2.47 were selected for the study. It was found that all the studied strains consume glucose, disaccharides (lactose, sucrose, maltose). B. longum 379 has more pronounced saccharo-lytic activity; in addition to the substrates listed above, this strain is able to consume mannose, trisaccharide melezitose, and raffinose. All strains are unable to ferment cellobiose (4-P-glucoside-glucose), trehalose disaccharide, rhamnose deoxysaccharide, aldoses (xylose, arabinose), glucosides (salicin, esculin), poly-hydric alcohols (glycerol, sorbitol, mannitol), gelatin.
It was found that the biochemical profiles of all the studied clones of probiotic strains are stable and correspond to the characteristics stated in the passports of the strains deposited in the State collection of pathogenic microorganisms, State collection of normal microflora microorganisms of G.N. Gabrichevsky Moscow Scientific Research Institute of Epidemiology and Microbiology, Russian national collection of industrial microorganisms (VKPM).
Results of whole genome sequencing of bacteria of the genus Bifidobacterium
One culture of each strain with known biochemical activity and the highest Score was se-
lected for whole genome sequencing: B. bifidum 1 - 2.37; B. bifidum 791 - 2.47; B. longum 379 - 2.33. The main characteristics of the strain genomes are presented in Table 4.
Whole genome sequences of the strains were deposited in the international database GenBank: B. bifidum 1, GenBank: NDXI00000000 (2017), B. bifidum 791, GenBank: LKUR00000000 (2015), B. longum 379, GenBank: LKUQ00000000 (2015).
Whole-genome sequence analysis for patho-genicity and antibiotic resistance determinants
Using the PathogenFinder and PlasmidFinder services, we found that the genomes of all studied strains did not contain pathogenicity determinants and integrated plasmids. Analysis of whole-genome sequences for the presence of antibiotic resistance genes was performed using two services: the ResFinder 3.2 program and the RAST genomic server. According to ResFinder 3.2, no antibiotic resistance determinants were detected in the genomes of all strains. RAST allowed us to identify molecular efflux pumps of the MATE family; in addition, a cytoplasmic protein that protects the ribosome from tetracycline exposure (tetW) was identified in B. longum 379 strain.
Analysis of genomic maps of the strains
Genomic maps of strains were obtained (Fig. 2, 3, 4). The genomic map allows us to graphically represent and visually evaluate the structure and nature of the genome of a microorganism.
An analysis of the obtained genomic maps revealed that all three strains have regions that are characterized by an increased or decreased level of GC bases. The presence of such regions may indicate that the genes located within them could have been introduced into the genome by horizontal transfer (Pinevich, 2009). These regions contain genes encoding metabolic enzymes (glycosyltransferases, xylanase, peptidase, sialidase, etc.) and TerB protein responsible for the resistance of the microorganism to harmful environmental factors, in particular to heavy metals.
Table 3
Biochemical properties of bifidobacteria under study - strains of probiotics producers
Strain IND URE GLU MAN LAC SAC MAL SAL XYL ARA GEL ESC GLY CEL MNE MLZ RAF SOR PHA TRE CAT
B. bifidum 1 - - + - + + +
B. bifidum 791 - - + - + + +
B. longum 379 - - + - + + + - - - - - - - + + + - - - -
* Note: IND - indole, URE - urease, GLU - glucose, MAN - mannitol, LAC - lactose, SAC - sucrose, MAL - maltose, SAL - salicin, XYL - xylose, ARA - arabinose, GEL - gelatin, ESC - esculin, GLY - glycerol, CEL - cellobiose, MNE - mannose, MLZ - melecytose, RAF - raffinose, SOR - sorbitol, PHA - rhamnose, TRE - trehalose, CAT - catalase.
Table 4
The main characteristics of the genomes of the studied strains of the genus Bifidobacterium
Strain Number of contigs Average coverage Genome size, bp GC composition, % Amount of CDS Number of tRNAs
B. bifidum 1, GenBank: NDXI00000000 13 385.0 2,198,027 62.7 1521 53
B. bifidum 791, GenBank: LKUR00000000 33 150.0 2,285,457 62.4 1769 52
B. longum 379, GenBank: LKUQ00000000 24 150.0 2,387,620 60.2 1903 71
Fig. 2. Genomic map of B. bifidum 1 strain obtained using the Proksee service (https://proksee.ca). GC content - content of GC bases (%), GC skew plus - regions with a high content of GC, GC skew minus - regions with a low content of GC, gene - genes, rRNA - determinants encoding ribosomal RNA, CDS - coding region, tRNA - transport RNA
Fig. 3. Genomic map of B. bifidum 791 strain obtained using Proksee service (https://proksee.ca). GC content - content of GC bases (%), GC skew plus - regions with a high content of GC, GC skew minus - regions with a low content of GC, gene - genes, rRNA - determinants encoding ribosomal RNA
Fig. 4. Genomic map of B. longum 379 strain obtained using Proksee service (https://proksee.ca). GC content - content of GC bases (%), GC skew plus - regions with a high content of GC, GC skew minus - regions with a low content of GC, gene - genes, rRNA - determinants encoding ribosomal RNA, CDS - coding region, tRNA - transport RNA, CDS - coding region, tRNA - transport RNA
Analysis of the phylogenetic relationship of the strains under study
To understand the phylogenetic relationships between the studied and other strains of these species, the whole genome sequences of which are deposited in the international GenBank database, dendrograms constructed using the BLAST program were analyzed. To date, 178 whole genome sequences of representatives of the B. bifidum species and 670 whole genome sequences of the B. longum species are deposited in the GenBank database. Analysis of the dendrogram revealed that B. bifidum 791 strain is part of a cluster that includes 17 strains (Fig. 5A). The most phylogenetically close to B. bifidum 791 strains are B. bifidum ICIS-176 (GenBank: JAJHZI000000000.1, depositor - Institute of Cellular and Intracellular Symbiosis UrB RAS, Orenburg, Russia), B. bifidum VKPM Ac-1784 (GenBank:
JACTOF000000000.1, depositor - Kurchatov Institute, Moscow, Russia), and B. bifidum ICIS-202 (GenBank: SSMS00000000.1, de positor - Institute of Cellular and Intracellular Symbiosis UrB RAS, Orenburg, Russia). All these strains were isolated from human feces and exhibit probiotic properties (Bukharin et al., 2019; Korzhenkovet al., 2020).
The B. bifidum 1 strain is part of a cluster that includes 16 strains (Fig. 5B).
Among the most phylogenetically close to B. bifidum 1 strain are B. bifidum NCTC13001 (GenBank Reference Sequence:
NZ_LR134344.1, depositor - Wellcome Sanger Institute, UK) isolated from infant feces, B. bifi-dum JCM 1255 (depositor - Graduate School of Frontier Sciences, University of Tokyo, Japan), also isolated from infant feces (Morita et al., 2019), and other variants of the B. bifidum JCM 1255 strain stored in various collections: B. bifidum ATCC 29251 (GenBank: AWSW00000000.1, depositor - Washington University Genome Sequencing Center, St. Louis, USA) and B. bifidum DSM 20456 (GenBank: JDUM00000000.1, depositor - School of Food Science and Engineering, China) (Vasiliev, 2017). In addition, the cluster of phylogenetically related strains includes B. bifidum UBBB-55 strain (GenBank: JADPYV000000000.1, depositor - Unique Biotech Limited, India; a probiotic strain with anticarcinogenic activity (Yenuganti
et al., 2021)), B. bifidum s-1 (GenBank: JA-CEIZ000000000.1, depositor - China Center of Industrial Culture Collection, China), B. bifidum BB-G90 (GenBank: JAGJCE000000000.1, depositor - Research and Development Center, China; a probiotic strain that is part of the probiotic to support athletes (Tavares-Silva et al., 2021)), B. bifidum LMG 11041 (GenBank: JGYO00000000.1, depositor - Life Sciences, University of Parma, Italy; probiotic strain isolated from infant feces). Thus, B. bifidum 1 and B. bifidum 791 strains belong to the clusters of probiotic strains isolated from human feces.
Figure 6 demonstrates a fragment of the dendrogram containing a cluster of strains phylogenetically close to B. longum 379. According to the dendrogram, this strain is part of a cluster that unites 12 strains; the most phylogenetically close to it are B. longum ICIS-206 (GenBank Reference Sequence: NZ_JAJBBZ000000000.1, depositor - Institute of Cellular and Intracellular Symbiosis UrB RAS, Orenburg, Russia; a probiotic strain isolated from the intestines of a healthy person (Ivanova et al., 2018)) and Bifidobacterium longum CCUG30698 (GenBank: CP011965.1, depositor - University College Cork, Ireland). It was found thatB. longum 379 is a member of a cluster formed by strains of the subspecies Bifidobacterium longum subsp. longum: B. longum subsp. longum CCUG30698 (GenBank: CP011965.1, University College Cork, Ireland), B. longum subsp. longum KCTC 3421 (GenBank Reference Sequence: NZ_CP071590.1, College of Health Science, Korea University, Korea), B. longum subsp. longum 296B (GenBank Reference Sequence: NZ_MLZK00000000.1, Life Sciences, University of Parma, Italy), B. longum subsp. longum VKPM Ac-1635 (GenBank Reference Sequence: NZ_JACTOE000000000.1, Kurchatov Institute), B. longum DS18_3 (GenBank Reference Sequence: NZ_QDJB01000174.1, Department of Molecular Biology, USA), B. longum subsp. longum BL-G301 (GenBank Reference Sequence: NZ_JAFKPI000000000.1, Basic Research Department, Research and Development Center, China), B. longum subsp. longum MCC10103 (GenBank Reference Sequence: NZ_SHSW01000009.1, depositor - Next Generation Science Institute, Japan), which allows us to attribute it to this subspecies.
Fig. 5. Dendrogram of B. bifidum 1 and B. bifidum 791 strains obtained using BLAST. Genomic BLAST file was downloaded from GenBank database
Fig. 6. Dendrogram of B. longum 379 strain obtained using BLAST. Genomic BLAST file was downloaded from GenBank database
Analysis of the level of genetic diversity of the strains
To assess the level of genetic diversity of B. bifidum and B. longum species, an analysis of pan- and core genomes of 695 strains of these species deposited in GenBank was carried out. Pangenome is the totality of all genes of the considered group of organisms (usually mono-phyletic), for which genetic diversity between closely related strains is possible. The core genome is genes present in all strains of the studied taxon.
To analyze the pan- and core genome of the B. bifidum species, graphs showing the dependence of the number of genes on the number of studied strains were constructed based on the genetic sequences of 95 strains (Fig. 7). It was found that the pangenome of B. bifidum contains 4952 protein-coding genes, while the core genome (genes present in > 95% of strains of the species) includes 1154 genes, and the accessory genome (genes present in < 95% strains) - 3798 genes. An analysis of the resulting graph allows us to conclude that the pangenome is open, which may indicate a significant role of horizontal gene transfer in the evolution of B. bifidum.
To analyze the level of genetic diversity of strains of the B. longum species, graphs showing the dependence of the number of genes on the number of studied strains were constructed based on the genetic sequences of 600 strains (Fig. 8).
It was found that the B. longum pangenome contains 15405 protein-coding genes, while the core genome (genes present in > 95% of strains of the species) includes 1082 genes, and the accessory genome (genes present in < 95% of strains) - 14323 genes. The pangenome of this species is also open, which may indicate a significant role of horizontal gene transfer in its evolution. Also noteworthy is the difference in the sizes of the accessory genomes of representatives of the B. bifidum and B. longum species, which may indicate a more pronounced metabolic activity of the B. longum strain.
Another method for assessing the phyloge-netic position of strains is the method of constructing a matrix of changes (SNP) from the
alignment of the amino acid sequences of strain core genome proteins with visualization in the form of a heat map (Fig. 9).
According to the color scale, species with similar protein sequences are colored dark blue; in the presence of dissimilar proteins, they have a shade from light blue to yellow. Analysis of the obtained heat map identified the strains with the closest sequences of proteins determined by the core genome genes: for B. bifidum 1 these are B. bifidum LMG 11041, B. bifidum NCTC13001, B. bifidum UBBB-55, B. bifidum s-1, B. bifidum BB-G90, the same strains, the phylogenetic relationship with which was found during the dendrogram analysis (Fig. 5). B. bifidum 791 strain deposited by us is a part of a unicolored cluster which includes B. bifidum ICIS-176, B. bifidum VKPM Ac-1784 strains, and the B. bifidum 791 strain of the same name, deposited by Institute of Cellular and Intracellular Symbiosis UrB RAS, Orenburg, Russia.
The analysis of the heat map constructed for strains of the B. longum species revealed that the studied B. longum 379 strain is included in a large cluster of strains with similar protein amino acid sequences; however, no strains with a high degree of identity were found (Fig. 10).
Thus, the pangenomes of both species are classified as open, which indicates a significant role of the process of horizontal gene transfer in the evolution of these species. The size of the B. bifidum pangenome was 4952 protein-coding genes, the core genome included 1154 genes; the size of the B. longum pan- and core genome was 15405 and 1082 genes, respectively. When analyzing the similarity of amino acid sequences of proteins encoded by the strains' core genome, it was found that B. bifidum 1 and B. bifidum 791 demonstrate a high degree of similarity with individual probiotic strains of this species. B. longum 379 is included in a large cluster that also contains a number of probiotic strains with similar amino acid sequences of proteins encoded by the core genome; however, no strains with a high degree of identity were found.
5DDÜ
25 50 75 POO
N umber of Genomes
Fig. 7. Graph showing the concept of B. bifidum bacterial pangenome
Fig. 8. Graph showing the concept of B. longum bacterial pangenome
Fig. 9. Heat map representing the percentage of average amino acid sequence identity of the core genomes of 95 strains of B. bifidum. The color key represents the percentage of identity among the strains, strains with few changes (blue), and those with more changes (red). Strains are grouped into dendrograms based on the average values of the series
Fig. 10. Heat map representing the percentage of average amino acid sequence identity of the core genomes of 600 strains of B. longum. The color key represents the percentage of identity among the strains, strains with few changes (blue), and those with more changes (red). Strains are grouped into dendrograms based on the average values of the series
Analysis of the metabolic potential and gly-cosyl hydrolase genes of the strains
The obtained genomic sequences were analyzed using the RAST (Rapid Annotation using Subsystems Technology) genomic server (http://rast.nmpdr.org). It was found that in all the strains studied, the most widely represented are the subsystems of protein and sugar metabolism; the subsystems of sugar metabolism of all strains include determinants of the phospho-ketolase pathway (fructose-6-phosphate phos-phoketolase pathway), whose products are lactic, acetic acids and ethanol.
The protein and sugar metabolism subsystems of the B. bifidum 1 strain consist of 225 and 175 determinants, respectively (Table 5). The strain has a low ability to metabolize mon-osaccharides but is active against di-, oligosac-charides, and amino sugars: chitin and N-acetylglucosamine. Exopolysaccharide synthesis genes were not found; however, there are determinants responsible for the formation of sortase-dependent pili (SrtA, GenBank: PDH97100.1, PDH97310.1), AP surface protein, as well as cell wall lipoproteins (Lgt, GenBank: PDH98440.1 and LspA, GenBank: PDH98074.1).
The protein and sugar metabolism subsystems of the B. bifidum 791 strain consist of 207 and 150 determinants, respectively (Table 5). In terms of its saccharolytic and other properties, B. bifidum 791 strain is close to B. bifidum 1 strain. Thus, the genome of strain B. bifidum 791 contains almost no determinants of mono-saccharide metabolism; however, there are genes responsible for the breakdown of more complex carbohydrates: di-, oligosaccharides, amino sugars, and starch. The genome contains genes encoding exopolysaccharide synthesis enzymes, including rhamnose synthesis genes located within contig 3 (GenBank LKUR01000023.1), determinants responsible for the formation of sortase-dependent pili (SrtA, GenBank: KYJ84870.1) and AP surface protein (GenBank: KYJ84871.1), as well as cell wall lipoproteins Lgt (GenBank: KYJ84380.1) and LspA (GenBank: KYJ85145.1).
The subsystems of protein and sugar metabolism of the B. longum 379 strain consist of 212
and 199 determinants, respectively (Table 5). The determinants responsible for the utilization of monosaccharides (xylose, ribose, arabinose), disaccharides (sucrose, maltose, lactose, raffi-nose, and phosphorylated oligosaccharide), amino sugars, and starch were found in the genome. Exopolysaccharide synthesis determinants are also presented: rhamnose synthesis genes located within contig 5 (GenBank: LKUQ01000020.1), capsular polysaccharide genes Wzb (tyrosine kinase) (GenBank: KYJ83195.1), Wzc (tyrosine phosphatase) KYJ83223.1, genes responsible for the formation of sortase-dependent pili - SrtA determinants (sortase A) (GenBank: KYJ83477.1) and AP (GenBank: KYJ83476.1), as well as lipoproteins - genes Lgt (GenBank: KYJ83617.1) and LspA (GenBank: KYJ77995.1).
Analysis of glycosyl hydrolase genes of the strains
The genome of bifidobacteria is specialized in the fermentation of a wide range of complex carbohydrates; therefore, a significant number of papers are devoted to the study of their gly-cosyl hydrolases (GH) - enzymes that catalyze the hydrolysis of glycosidic bonds in carbohydrate molecules, leading to the appearance of smaller molecules. The analysis of glycosyl hydrolases of the studied strains identified several groups of these enzymes. The first group was found in the genomes of all three strains: GH2, GH13 (enzymes that break a-glucosidic bonds, such as a-amyl-ases), GH20 (P-hexosaminidase), GH3 (P-glu-cosidases, P-xylosidases, N-acetylhex-ominidase, which contribute to mucin glycan degradation), GH32 (invertase), GH36 (a-ga-lactosidase), GH42 (P-galactosidase), GH43, GH51 (a-L-arabinofurosidase (plant fiber utilization)), GH7 (endo-P-1,4-arabinogalactan galactanase), GH101 (endo-a-N-acetylgalac-tosominidase (GMO and mucin degradation)), GH129 (a-N-acetylgalactosominase (mucin degradation)). The presence of these enzymes reflects the basic ability of the studied strains of bifidobacteria to consume galactooligosac-charides, including breast milk, plant fibers, and mucin glycans (Table 6).
Glycosyl hydrolases of the second group were found only in the genomes of B. bifidum 1 and B. bifidum 791: GH1, GH29 (a-fucosidases), GH110 (a-1,3 -gal actosidase), GH123 (P-N-acetylgalactose minidase, glycosphingolipid P-N-acetylgalactosominidase (degradation of mucin glycans)). The genome of B. bifidum 791 contains GH84 (hyaluronic glucosaminidase, P-N-acetyl hexosominidase), GH95 (a-1,2-L-fuco-sidase), GH33 (sialidase), enzymes also associated with the degradation of mucin glycans.
Glycosyl hydrolases of the third group were present only in the B. longum 379 ge
nome: GH31 (a-D-xyloside xylohydrolase), GH5, GH30, GH12 (xylosidases), GH121, GH127, GH146 (3.2.1.185 P-L-arabino-furanosidase). All these enzymes belong to hemicellulases and are associated with the utilization of plant pol ysaccharides - plant fibers and mucus. Compared to B. bifidum 1 and B. bifidum 791, the genome of B. longum 379 also contains a larger number of enzymes of the GH13 groups - amylases (8) and GH43 - arabinofurazidases (10), which also play a crucial role in the breakdown and use of plant polysaccharides.
Table 5
Functional annotation of strains using RAST
Subsystem name, number of genes Strains
B. bifidum 1 B. bifidum 791 B. longum 379
Carbohydrates 175 150 199
Protein metabolism 225 207 212
Amino Acid and Derivatives 225 198 207
DNA Metabolism 80 66 67
Fatty acids, Lipids end Isoprenoids 40 27 25
Cofactors, Vitamins, Prosthetic Groups, Pigments 77 73 96
Cell Wall 63 70 65
Table 6
Representatives of glycosyl hydrolase families in the genomes of the studied strains
Family name according to CAZy classification The number of determinants encoding an enzyme in the strain genome; enzyme classification according to Enzyme Classification (EC)
B. bifidum 1 B. bifidum 791 B. longum 379
GH2 1 ß-galactosidase (EC 3.2.1.23) 3 ß-galactosidase (EC 3.2.1.23) 1 ß-galactosidase (EC 3.2.1.23)
GH3 1 ß-glucosidases (EC 3.2.1.21) 3 ß-glucosidase (EC 3.2.1.21)
GH13 2 phosphate a-maltosyl- transferase (EC 2.4.99.16) 3 phosphate a-maltosyltransferase (EC 2.4.99.16) a-glucosidase (EC 3.2.1.20) 8 isomaltulose synthase oligo-a-glucosidase (EC 3.2.1.20) a-glucosidase (EC 3.2.1.20) glucodextranase (EC 3.2.1.68) isoamylase (EC 3.2.1.68)
Continuation of the table 6
Family name according to CAZy classification The number of determinants encoding an enzyme in the strain genome; enzyme classification according to Enzyme Classification (EC)
B. bifidum 1 B. bifidum 791 B. longum 379
4-a-glucanotransferase (EC 2.4.1.25) branching enzyme (EC 2.4.1.18) isomaltulose synthase (EC 5.4.99.11) starch synthase (malto-syl-transferring) (EC 2.4.99.16) sucrose phosphorylase (EC 2.4.1.7)
GH20 1 1 ß-hexosaminidase (EC 3.2.1.52) 1 ß-hexosaminidase (EC 3.2.1.52) lacto-N-biosidase mucine (EC 3.2.1.140) 1 P-hexosaminidase (EC 3.2.1.52)
GH29 1 dextranase (EC 3.2.1.11) 1 dextranase (EC 3.2.1.11) -
GH30 1 endo-P-1,4-xylanase (EC 3.2.1.8);
GH31 2 a-D-xyloside xylohydrolase (EC 3.2.1.177)
GH32 1 invertase (EC 3.2.1.26); 1 invertase (EC 3.2.1.26); 2 invertase (EC 3.2.1.26);
GH33 1 sialidase (EC 3.2.1.18) -
GH36 1 a-galactosidase (EC 3.2.1.22) 1 a-galactosidase (EC 3.2.1.22) 1 a-galactosidase (EC 3.2.1.22)
GH42) 1 (EC 3.2.1.23) 2 (EC 3.2.1.23) 2 (EC 3.2.1.23)
GH43 1 ß-xylosidase (EC 3.2.1.37) 1 ß-xylosidase (EC 3.2.1.37) 10 non-reducing end a-L-arabinofuranosidase (EC 3.2.1.37) xylan 1,4-P-xylosidase (EC 3.2.1.145) galactan 1,3-P-galactosidase (EC 3.2.1.8) endo- 1,4-P-xylanase (EC 3.2.1.37)
GH51 2 endoglucanase (EC 3.2.1.4); endo-ß-1,4-xylanase (EC 3.2.1.8); ß-xylosidase 1 endoglucanase (EC 3.2.1.4) 5 a-L-arabinofuranosidase (EC 3.2.1.55)
Continuation of the table 6
Family name according to CAZy classification The number of determinants encoding an enzyme in the strain genome; enzyme classification according to Enzyme Classification (EC)
B. bifidum 1 B. bifidum 791 B. longum 379
GH53 1 arabinogalactan endo-ß-1,4-galactanase (EC 3.2.1.89)
GH77 1 amylomaltase or 4-a-glu- canotransferase (EC 2.4.1.25) 1 amylomaltase or 4-a-glu-canotransferase (EC 2.4.1.25) 2 arabinogalactan endo-ß-1,4-galactanase (EC 2.4.1.25)
GH84 2 acetylhexosaminidase (EC 3.2.1.52)
GH95 1 a- 1,2-L-fucosidase (EC 3.2.1.63)
GH101 1 endo-a-V- acetylgalactosaminidase (EC 3.2.1.97) 1 endo-a-V- acetylgalactosaminidase (EC 3.2.1.97) 1 endo-a-N- acetylgalactosaminidas e (EC 3.2.1.97)
GH109 1 a-N- acetylgalactosaminidase (EC 3.2.1.49); 1 a-N-acetylgalactos- aminidase (EC 3.2.1.49);
GH110 1 a-1,3 -galactosidase (EC 3.2.1.-) 1 a-1,3 -galactosidase (EC 3.2.1.-)
GH112 1 lacto-N-biose Phosphorylase or galacto-N-biose phosphorylase (EC 2.4.1.211); 2 lacto-N-biose Phosphorylase or galacto-N-biose phosphorylase (EC 2.4.1.211); 1 lacto-N-biose Phosphorylase or galacto-N-biose phosphorylase (EC 2.4.1.211);
GH121 1 ß-L-arabinobiosidase (EC 3.2.1.-)
GH120 1 3.2.1.37 ß-xylosidase (EC 3.2.1.37)
GH123 1 ß-N- acetylgalactosaminidase (EC 3.2.1.53); 1 ß-N- acetylgalactosaminidase (EC 3.2.1.53);
GH127 2 non-reducing end ß-L-arabinofuranosidase (EC 3.2.1.185)
The end of the table 6
Family name according to CAZy classification The number of determinants encoding an enzyme in the strain genome; enzyme classification according to Enzyme Classification (EC)
B. bifidum 1 B. bifidum 791 B. longum 379
GH129 1 a-N- acetylgalactosaminidase (EC 3.2.1.49) 1 a-N- acetylgalactosaminidase (EC 3.2.1.49) 1 a-N- acetylgalactosamini-dase (EC 3.2.1.49)
GH136 lacto-N-biosidase (EC 3.2.1.140); — 1 3.2.1.140
GH146 1 ß-L-arabino-furanosidase (EC 3.2.1.185)
Analysis of the ability of strains to synthesize neurometabolites
Analysis of the genomes of the studied strains in the genomes of B. bifidum 791 and B. longum 379 revealed the key enzymes for the synthesis of neurometabolites - tryptophan and folic acid. Thus, the B. bifidum 791 genome contains genes that determine the synthesis of a and P subunits of tryptophan synthase (GenBank: KYJ84379.1, KYJ84378.1), an enzyme responsible for the synthesis of tryptophan, and dihydropteroate synthase (GenBank: KYJ84132.1), responsible for folic acid synthesis. The genome of B. longum 379 also contains tryptophan synthase, a and P subunits (GenBank: KYJ83618.1, KYJ83619.1) and dihydropteroate synthase (GenBank: KYJ81979.1).
Analysis of the ability of strains to synthesize bacteriocins
The genomes of all studied bifidobacteria were analyzed for the presence of genes that determine bacteriocins. The B. bifidum 791 genome (contig 28; GenBank: LKUR01000021.1) contains determinants responsible for the synthesis of class I lantibiotics: lasso peptide, a ribosomal-pro-duced peptide, and bacteriocin flavucin
(Fig.11).
Analysis of the operon responsible for the synthesis of the lasso peptide revealed that it consists of the gene responsible for the synthesis of the linear precursor (core peptide) and the LasC synthetase, consisting of 3 subunits,
which determines the conformational structure of the final product - the transformation of the linear peptide into the lasso peptide, in which the C-terminal part of the molecule is «threaded» through the N-terminal macrolac-tam ring. The operon also includes genes for ABC transporters responsible for peptide secretion (Fig. 11A). The amino acid sequence of the B. bifidum 791 lasso peptide was determined: LIMKQYIAPTVETIASFREATNGLWFGK-YVDIGGAKAPFPWGSN.
Analysis of the similarity of protein sequences using BLAST showed that the B. bifidum 791 lasso peptide is highly similarity to other lasso peptides of probiotic strains of bifidobacteria (Fig. 12).
Flavucin is an antimicrobial peptide similar in structure to nisin, a bacteriocin synthesized by Lactococcus lactis. An analysis of the op-eron responsible for flavucin synthesis revealed that the gene responsible for the synthesis of the core protein is flanked by the LanC (Lantibiotic biosynthesis protein) modification determinant and several ABC transporters (Fig. 11C). The amino acid sequence of B. bifidum 791 flavucin was determined: MATFDLDTHVEETEG-DARPQITSKFMCTPGCPTGGLACFTSQC-TKGCSFTGGSKH.
When analyzing the similarity of protein sequences using BLAST, it was found that B. bi-fidum 791 flavucin has a high similarity with other bacteriocins of the lantibiotic class -gallidermin and nisin (Fig. 13).
Fig. 11. Operons responsible for the synthesis of the lasso peptide (A) and flavucin (B) of B. bifidum 791 (data obtained using the BAGEL 4.0 genomic server)
Fig. 12. Phylogenetic tree of amino acid sequences of lasso peptides of bacteria of the genus Bifidobacterium using the BLAST program
Fig. 13. Phylogenetic tree of amino acid sequences of lantibiotics of bacteria of the genus Bifidobacterium using the BLAST program
Discussion
It was found that probiotic bifidobacteria Bifidobacterium bifidum 1, Bifidobacterium bi-fidum 791, Bifidobacterium longum 379 have morphological, tinctorial, cultural, and stable biochemical properties typical for these strains. For the first time, direct profiling of bacterial proteins using MALDI TOF mass spectrometry revealed a list of masses of ionized proteins (peaks) typical for each of the three strains. The B. bifidum 791 strain is characterized by peaks corresponding to the following masses m/z: 12027, 10445, 10138, 9841, 9502, 9285, 8927, 6546, 6390, 6014, 4921, 3399, 3273, 2304, 2186; for strain B. bifidum 1 the following peaks are specific: 13183, 10121, 8716; for strain B. longum 379: 13124, 11977, 9831, 9261, 8790, 8316, 8193, 6567, 6305, 5991, 4916, 4631, 4395, 3283, 3153.
As a result of the analysis of whole genome sequences of bifidobacteria, it was found that the genomes of B. bifidum 1, B. bifidum 791, and B. longum 379 strains do not contain pathogenicity genes and integrated plasmids. Efflux pumps of the MATE family are antiporter membrane transport proteins that export drug compounds from the bacterial cell and do not belong to antibiotic resistance determinants. The tetW gene encoding a protein that protects the ribosome from the action of tetracycline is located within the sixth contig (LKUQ01000021.1); an analysis of its genomic context showed that this gene is not associated with the determinants responsible for the mobile transfer, and therefore does not pose a threat in terms of transmission.
The analysis of genomic maps of strains revealed that genes associated with parallel transfer encode metabolic enzymes (glycosyltrans-ferases, xylanase, etc.), proteins responsible for resistance to harmful environmental factors, such as heavy metals (tellurite resistance protein, TerB) and proteins with an unknown functional role (hypothetical protein). The analysis of phylogenetic relationships of B. bifidum 1, B. bifidum 791, and B. longum 379 with 848 strains of these species, whole genome sequences are deposited in GenBank, revealed that the studied strains are part of phylogenetic
clusters formed by probiotic strains, including foreign strains-producers of probiotic medicines and foodstuffs.
Pan- and core genomes of all B. bifidum (n = 95) and B. longum (n = 600) strains presented in GenBank were analyzed by the number of protein-coding genes. The pangenome of B. bifidum contains 4952 genes, the core genome -1154 genes; the pangenome and core genome of B. longum contain 15405 and 1082 genes, respectively. The pangenomes of both species are classified as open, indicating a significant role of horizontal gene transfer in the evolution of these species. An analysis of the similarity of the amino acid sequences of proteins encoded by the core genome of the strains revealed that B. bifidum 1 and B. bifidum 791 demonstrate a high degree of similarity with individual probiotic strains of this species, including probiotic-producing strains. B. longum 379 is included in a large cluster that also contains a number of probiotic strains with similar amino acid sequences of proteins encoded by the core genome; however, strains with a high degree of identity were not found.
Analysis of the genetically determined metabolic potential of B. bifidum 1, B. bifidum 791, and B. longum 379 allowed us to reveal that the subsystems of protein and sugar metabolism are the most widely represented in them. It was shown that all strains possess determinants of the phosphoketolase pathway and are able to consume di-, oligosaccharides, and amino sugars. The products of active carbohydrate breakdown are lactic, acetic acids, and ethanol, which are among the most important biologically active compounds that provide slight acidification of the intestinal cavity, promote better absorption of electrolytes and inhibit the growth of pathogenic and opportunistic microorganisms. A high level of lactate stimulates the protrusion of macrophages (penetration of their processes from the mucosa into the intestinal cavity) and determines the immunoregulatory properties of bifidobacteria, while acetate, a valuable short-chain fatty acid, performs important energy tasks by being absorbed into the bloodstream and entering the cells of various organs and tissues, reduces the level of toxic metabolites and
carcinogens, normalizes the motility of the gastrointestinal tract, reduces the formation of ketones (Morita et al., 2019; Kornienko, 2016).
The genome of bifidobacteria is specialized in the fermentation of a wide range of complex carbohydrates; therefore, a significant number of papers are devoted to the study of their gly-cosyl hydrolases, enzymes that catalyze the hydrolysis of glycosidic bonds in carbohydrate molecules, leading to the appearance of smaller molecules. Glycosyl hydrolases (GH) ensure the utilization of disaccharides, plant polysac-charides, breast milk oligosaccharides, and gly-cans of intestinal mucus, and statistically significant differences in the spectrum of these enzymes in different species of the genus Bifidobacterium were revealed, which reflects the specifics of their adaptation to the ecological niche occupied - the human colon (Schell et al., 2002; Turroni et al., 2010; Chaplin, 2015; Morita et al., 2019; Fushinobu et al., 2021; Kelly et al., 2021). Analysis of the glycosyl hydrolase spectrum revealed that B. bifidum 1 and B. bifidum 791 strains have a pronounced ability to consume glycans of intestinal mucus. Their genomes contain enzymes for the degradation of mucin oligosaccharides: fucosidases, P-N-acetylgalactosaminidase, lacto-N-bio-sidase, hyaluronoglucosaminidase. A complex of specialized enzymes that allow efficient utilization of plant glycans, including xylo- and arabino-oligosaccharides, was identified in the genome of B. longum 379. The results obtained reflect the adaptation of these strains to the conditions of existence under a deficiency of simple carbohydrates in the lower intestine. Such a «distinction» in the choice of carbohydrate sources allows these strains of bifidobacteria to effectively coexist in vivo, avoiding competition for nutrient substrates. In addition, the complex of active bacterial hydrolases ensures the implementation of the probiotic properties of the strains: colonization resistance, antagonistic activity against the PM and OM, active symbiotic digestion, and, consequently, the most efficient absorption of food by the macro-organism.
It was also shown that B. longum 379 and B. bifidum 791 strains contain in the genome de-
terminants of the synthesis of exopolysaccha-rides, sortase-dependent pili, and cell wall lipo-proteins (LpAs). Exopolysaccharides of probi-otic bacteria, sortase-dependent pili, and lipo-proteins can physically prevent the adhesion of viruses; in addition, exopolysaccharides of bifidobacteria protect cells from aggressive secretions of the gastrointestinal tract, have a pronounced antibacterial effect, and are also used by other members of the intestinal microbiota as a nutrient substrate (Ermolenko et al., 2003; El Kfoury et al., 2017; Kang et al., 2015).
Determinants encoding enzymes of trypto-phan and folic acid synthesis, the most important neurometabolites with a pronounced thymoleptic effect (correction of increased anxiety, depression, and cognitive decline), were found in the genomes of the strains under study. Previously, researchers have already obtained evidence of the ability of bifidobacteria to increase the level of tryptophan in the blood of laboratory animals, to alleviate their condition under stress, that is, to have a thymoleptic effect (Oleskin & Shenderov, 2016). Tryptophan produced by bacteria and the tryptamine formed as a result of its decarboxylation can be delivered with the bloodstream to the brain and act as precursors of monoamine neurotransmitters, including serotonin (Oleskin & Shenderov, 2019). Serotonin deficiency causes pronounced brain disorders such as cognitive decline, increased anxiety, and depression.
Recently, bacteriocins synthesized by representatives of the normal human microbiota and probiotic-producing strains, including bacteria of the Bifidobacterium genus, have attracted the attention of researchers. Bacteriocins are pep-tides with broad-spectrum antimicrobial activity; the ability to synthesize them is strain-specific (Zaslavskaya et al., 2019). It is known that strains capable of synthesizing bacteriocins have an ecological advantage over other strains under in vivo conditions, and bacteriocins of strains of normal microbiota are involved in the formation of "joint immunity" of the macroor-ganism (Zaslavskaya et al., 2019). In the B. bifidum 791 genome, genes responsible for the synthesis of the lasso peptide and flavucin, bac-teriocins belonging to class I lantibiotics with a
wide spectrum of antimicrobial activity, which was previously proven empirically, were found (Solovyeva et al., 2012; Novik, 2006). Lantibi-otics are antimicrobial substances containing the typical thioether amino acids lanthionine or methyllanthionine; most lantibiotics are synthesized on ribosomes and undergo post-transla-tional modification. According to modern scientific data, lasso peptides have a wide range of antimicrobial and antiviral activity; they are active against bacteria of the genera Salmonella, Shigella, E. coli, P. aeruginosa, etc. The activity of lasso peptides against viruses is associated with the blocking of viral enzymes and receptors (van Heel et al., 2016; Lu et al., 2021;
Tiwari et al., 2020; Alvarez-Sieiro et al., 2016; Maksimov et al., 2012; Cheng & Hua, 2020). Flavucin is also known to have a wide spectrum of antimicrobial activity; there is data on the ability of structurally similar bacteriocins to inhibit all stages of viral replication, block their receptors, and prevent adsorption on eukaryotic cells (Lu et al., 2021). The presence of lasso peptide and flavucin synthesis determinants in the genome of B. bifidum 791 strain is associated with its activity against the epidemic influenza A/Lipetsk/1V/2018 virus (H1N1) and the highly pathogenic avian influenza A/common gull/Saratov/1676/2018 virus (H5N6) (Soloveva et al., 2021; Tochilina et al., 2022).
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